Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH, 43210, USA.
Arch Toxicol. 2018 Mar;92(3):1283-1293. doi: 10.1007/s00204-017-2135-2. Epub 2017 Nov 28.
Low-dose fluoride is an effective caries prophylactic, but high-dose fluoride is an environmental health hazard that causes skeletal and dental fluorosis. Treatments to prevent fluorosis and the molecular pathways responsive to fluoride exposure remain to be elucidated. Previously we showed that fluoride activates SIRT1 as an adaptive response to protect cells. Here, we demonstrate that fluoride induced p53 acetylation (Ac-p53) [Lys379], which is a SIRT1 deacetylation target, in ameloblast-derived LS8 cells in vitro and in enamel organ in vivo. Here we assessed SIRT1 function on fluoride-induced Ac-p53 formation using CRISPR/Cas9-mediated Sirt1 knockout (LS8) cells or CRISPR/dCas9/SAM-mediated Sirt1 overexpressing (LS8) cells. NaF (5 mM) induced Ac-p53 formation and increased cell cycle arrest via Cdkn1a/p21 expression in Wild-type (WT) cells. However, fluoride-induced Ac-p53 was suppressed by the SIRT1 activator resveratrol (50 µM). Without fluoride, Ac-p53 persisted in LS8 cells, whereas it decreased in LS8. Fluoride-induced Ac-p53 formation was also suppressed in LS8 cells. Compared to WT cells, fluoride-induced Cdkn1a/p21 expression was elevated in LS8 and these cells were more susceptible to fluoride-induced growth inhibition. In contrast, LS8 cells were significantly more resistant. In addition, fluoride-induced cytochrome-c release and caspase-3 activation were suppressed in LS8 cells. Fluoride induced expression of the DNA double strand break marker γH2AX in WT cells and this was augmented in LS8 cells, but was attenuated in LS8 cells. Our results suggest that SIRT1 deacetylates Ac-p53 to mitigate fluoride-induced cell growth inhibition, mitochondrial damage, DNA damage and apoptosis. This is the first report implicating Ac-p53 in fluoride toxicity.
低剂量氟化物是一种有效的龋齿预防剂，但高剂量氟化物是一种环境健康危害，会导致骨骼和牙齿氟中毒。预防氟中毒的治疗方法和对氟暴露有反应的分子途径仍有待阐明。此前我们表明，氟化物激活 SIRT1 作为一种适应性反应来保护细胞。在这里，我们证明氟化物诱导了 LS8 细胞中的 p53 乙酰化（Ac-p53）[Lys379]，这是 SIRT1 去乙酰化的靶点，体外在釉质器官中也有这种现象。在这里，我们使用 CRISPR/Cas9 介导的 Sirt1 敲除（LS8）细胞或 CRISPR/dCas9/SAM 介导的 Sirt1 过表达（LS8）细胞评估 SIRT1 功能对氟化物诱导的 Ac-p53 形成的影响。在野生型（WT）细胞中，NaF（5 mM）诱导 Ac-p53 形成并通过 Cdkn1a/p21 表达增加细胞周期停滞。然而，氟化物诱导的 Ac-p53 被 SIRT1 激活剂白藜芦醇（50 µM）抑制。没有氟化物，Ac-p53 在 LS8 细胞中持续存在，而在 LS8 细胞中减少。氟化物诱导的 Ac-p53 形成也在 LS8 细胞中受到抑制。与 WT 细胞相比，氟化物诱导的 Cdkn1a/p21 表达在 LS8 中升高，这些细胞对氟化物诱导的生长抑制更敏感。相比之下，LS8 细胞的抗性显著增强。此外，氟化物诱导的细胞色素 c 释放和 caspase-3 激活在 LS8 细胞中受到抑制。氟化物诱导 WT 细胞中 DNA 双链断裂标志物 γH2AX 的表达增加，在 LS8 细胞中增加，但在 LS8 细胞中减弱。我们的结果表明，SIRT1 去乙酰化 Ac-p53 以减轻氟化物诱导的细胞生长抑制、线粒体损伤、DNA 损伤和细胞凋亡。这是第一个表明 Ac-p53 参与氟化物毒性的报告。
